Endo-1,4-β-glucanase gene and its use in plants

ABSTRACT

The present invention provides a method for reducing fruit softening and cell wall polysaccharide degradation by inhibiting endo-1,4-β-glucanase activity using antisense DNA constructions.

This invention was made with Government support under Grant Nos.CRCR-87-1-2525 and CRCR-87-1-2526 awarded by the U.S. Department ofAgriculture. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods for reducing fruitsoftening. In particular, it relates to methods for reducing fruitsoftening and cell wall polysaccharide degradation by inhibiting theactivity of endo-1,4-β-glucanase.

2. Information Disclosure

Ripening, the final phase of fruit development, involves a number ofdramatic metabolic changes in fruit tissue. An important aspect of theripening process is fruit softening, which is thought to resultprimarily from modifications of the cell wall. Many subtle changes inmetabolic activity are involved in this response.

The prior art discloses ripening-impaired mutants, such as the rinmutant which have been used to study fruit ripening. Tigchelaar Hortic.Sci., 13:508-513, 1978. The use of these mutants to specifically controlfruit softening has met with limited success, however, because of thepleiotropic nature of these mutations.

An increase in the activity of polygalacturonase, an enzyme responsiblefor the degradation of pectin, has been correlated with fruit softening.Recombinant constructs have been prepared containing a plant promoterlinked to polygalacturonase cDNA in the antisense direction. Theseconstructs have been inserted into tomato to inhibit the activity ofthis enzyme in ripening fruit. Smith et al., Nature, 334:724-726, 1988;Sheehy et al., Proc. Nat. Acad. Sci., 85:8805-8809, 1988; Hiatt et al.,U.S. Pat. No. 4,801,340; Bridges et al., EPO Publication No. 0,271,988.Although these constructs have been shown to inhibit polygalacturonaseactivity, an effect on fruit softening has not been shown. Smith et al.,Plant Mol. 14:369-379, 1990.

Endo-1,4-β-glucanase is another enzyme thought to be involved in fruitsoftening. It is known to degrade the major hemicellulosic polymer,xyloglucan. Hatfield and Nevins, Plant and Cell Physiol., 27:541-552,1986. The cDNA and gene encoding endo-1,4-β-glucanase have been clonedfrom avocado (Christoffersen et al., Plant Molec. Biol., 3:385, 1984)and bean (Tucker et al., Plant Physiol., 88:1257, 1988).

SUMMARY OF THE INVENTION

The present invention relates to a method of reducing fruit softeningand inhibiting the degradation of cell wall polymers comprising,introducing into a plant an expression cassette having a plant promotersequence operably linked to a DNA subsequence of at least 20 base pairsderived from a DNA sequence encoding endo-1,4-β-glucanase, the DNAsubsequence being linked to the promoter sequence in the oppositeorientation for expression. The promoter can be either inducible orconstitutive. If inducible, it is preferably derived from the tomato E8gene. If constitutive, it is preferably the 35S promoter of cauliflowermosaic virus.

The method can be modified by introducing into a plant an expressioncassette as described above plus an expression cassette having a plantpromoter sequence operably linked to a DNA subsequence of at least 20base pairs derived from a DNA sequence encoding polygalacturonase, theDNA subsequence being linked to the promoter sequence in the oppositeorientation for expression.

The preferred plant for the method is tomato. The expression cassettecan be introduced into the plant by any in vitro technique, preferablyusing Agrobacterium. The expression cassette can also be introduced intothe plant by a sexual cross.

The present invention also provides a method of inhibitingendo-1,4-β-glucanase activity comprising, introducing into a plant anexpression cassette having a plant promoter sequence operably linked toa DNA subsequence of at least 20 base pairs derived from a DNA sequenceencoding endo-1,4-β-glucanase, the DNA subsequence being linked to thepromoter sequence in the opposite orientation for expression. Byinhibiting the enzyme, cell wall polysaccharide degradation can beinhibited. The preferred embodiments as described above apply to thismethod, as well, except that an expression cassette comprisingpolygalacturonase antisense DNA is not used.

The present invention further provides an expression cassette comprisinga plant promoter sequence operably linked to a DNA subsequence of atleast 20 base pairs derived from a DNA sequence encodingendo-1,4-β-glucanase, the DNA subsequence being linked to the promotersequence in the opposite orientation for expression. The promoter can beinducible, typically the E8 promoter, or constitutive, typically derivedfrom cauliflower mosaic virus.

A plant, preferably tomato, is also provided that contains an expressioncassette having a plant promoter sequence operably linked to a DNAsubsequence of at least 20 base pairs derived from a DNA sequenceencoding endo-1,4-β-glucanase, the DNA subsequence being linked to thepromoter sequence in the opposite orientation for expression.

The present invention further provides a DNA sequence which isuninterrupted, which encodes endo-1,4-β-glucanase, and which is flankedon at least one side by non-wild type DNA. The DNA sequence is typicallya cDNA sequence derived from tomato.

Further, an expression cassette is provided which comprises a promotersequence operably linked to a DNA sequence which is uninterrupted andwhich encodes endo-1,4-β-glucanase. The DNA sequence is typically a cDNAsequence derived from tomato. The promoter sequence function in bothprokaryotes and eukaryotes.

The present invention also provides a method of isolating from a plant aDNA sequence encoding endo-1,4-β-glucanase comprising, probing a DNAlibrary prepared from plant tissue with oligonucleotide probescomprising a conserved sequence from the endo-1,4-β-glucanase cDNA. TheDNA library can be either a genomic or cDNA library. The preferredconserved sequences are: ##STR1##

Finally, a DNA construct is provided comprising a promoter sequenceoperably linked to a DNA sequence encoding a signal peptide from tomatoendo-1,4-β-glucanase, the DNA sequence being joined to other than asequence encoding mature tomato endo-1,4-β-glucanase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-2 illustrate the construction of the pMLJ1:E8antiPG/CL andpMLJ1:CamVantiPG/CL vectors, respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENT

An improved method of reducing fruit softening in various agronomicallyimportant plant species is provided. The method comprises transforming aplant cell with an expression cassette having a plant promoter operablylinked to endo-1,4-β-glucanase (glucanase) DNA in the oppositeorientation for normal expression. In addition, a second expressioncassette can also be introduced that comprises polygalacturonase DNA inthe opposite orientation for normal expression. The glucanase cDNA canalso be inserted in correct orientation for expression of the gene inplant or bacterial cells. Also provided are nucleic acid probescomprising conserved regions of the endo-1,4-β-glucanase gene which canbe used to isolate the gene from any plant species. The cDNA sequenceprovided by this invention can be used to construct vectors capable ofexpressing fusion proteins comprised of the glucanase signal peptidefused to any foreign gene. This provides for the secretion of foreigngene products from the plant cell.

Control of the rate of fruit softening during the ripening process is oftremendous economic importance. In the case of tomatoes, inhibition offruit softening allows fresh market tomatoes to remain firm whileripening on the vine. Vine ripened tomatoes have better flavor and colordevelopment then those that are picked while green. Control of fruitripening may also improve fruit quality by increasing pathogenresistance. These properties allow for longer shelf and shipping life ofthe tomato fruit. Inhibition of cell wall degradation may also enhancethe processing characteristics of the tomato fruit by increasing fruitviscosity.

The present invention provides a method for reducing fruit softening byinhibiting the activity of glucanase in various agronomically importantspecies. In the exemplified case, cDNA from the tomato glucanase gene isused to create expression cassettes comprising antisense DNA to controlthe activity of the gene during fruit ripening.

Recombinant DNA techniques are used to introduce the antisense cDNAsequence into a suitable vector which is subsequently used to transforma suitable host cell. In the exemplified case, Agrobacterium tumefaciensis used as a vehicle for transmission of the cDNA to the ultimate host,the tomato cell. A plant regenerated from the transformed celltranscribes the antisense cDNA which inhibits activity of the enzyme. Inplant cells, it has been shown that cDNA inhibits gene expression bypreventing the accumulation of mRNA which results in decreased levels ofthe protein encoded by the gene, Sheehy et al., supra.

The following descriptions will detail various methods available tointroduce and express foreign DNA sequences in plant cells. Specificexamples of preferred methods are also described.

In summary, the manipulations necessary to prepare antisense glucanasecDNA and introduce it into a plant cell involve 1) isolating mRNA fromripe fruit, 2) preparing cDNA from the mRNA, 3) screening the cDNA forthe desired sequence, 4) linking a plant promoter to the desired cDNA inthe opposite orientation for expression of the glucanase gene, 5)transforming suitable host plant cells, and 6) selecting andregenerating cells which transcribe the inverted sequence.

I. General Methods

Generally, the nomenclature used hereafter and the laboratory proceduresin recombinant DNA technology described below are those well known andcommonly employed in the art. Standard techniques are used for cloning,DNA and RNA isolation, amplification and purification. Generallyenzymatic reactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like are performed according to the manufacturer'sspecifications. These techniques and various other techniques aregenerally performed according to Sambrook et al., Molecular Cloning--ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989. The manual is hereinafter referred to as "Sambrook". Othergeneral references are provided throughout this document. The procedurestherein are believed to be well known in the art and are provided forthe convenience of the reader. All the information contained therein isincorporated herein by reference.

II. Preparation of endo-1,4-β-glucanase cDNA.

To prepare glucanase cDNA, mRNA from ripe fruit is first isolated.Eukaryotic mRNA has at its 3' end a string of adenine nucleotideresidues known as the poly-A tail.

Short chains of oligo d-T nucleotides are then hybridized with thepoly-A tails and serve as a primer for the enzyme reverse transcriptase.This enzyme uses RNA as a template to synthesize a complementary DNAstrand. A second DNA strand is then synthesized using the first cDNAstrand as a template. Linkers are added to the double-stranded cDNA forinsertion into a plasmid or λ phage vector for propagation in E. coli.

Identification of clones harboring the desired cDNA is performed byeither nucleic acid hybridization or immunological detection of theencoded protein, if an expression vector is used. The bacterial coloniesare then replica plated on nitrocellulose filters. The cells are lysedand probed with either oligonucleotides complimentary to the desiredcDNA or with antibodies to the desired protein.

The nucleotide sequence of the cDNA encoding tomato endo-1,4-β-glucanaseis provided in Table I, below. The cDNA was deposited with the AmericanType Culture Collection, under the provisions of the Budapest Treaty onApr. 20, 1990, and has Accession No. 68312. The sequence may be used inany of a number of ways. Fragments of the sequence can be used as probesto identify glucanase genes in genomic or cDNA libraries prepared fromother plant species.

The cDNA can be inserted in the antisense direction into expressioncassettes to inhibit the expression of the glucanase gene in plantcells. The cDNA sequence, itself, can also be inserted in an expressioncassette for expression in bacteria or plant cells. Insertion of theexpression cassette in bacteria is useful for biomass conversion ofplant tissues to ethanol or methanol.

The sequence provided can also be used for expression of fusion proteinscomprised of a portion of the glucanase enzyme fused to another protein.Of particular interest is the transit peptide sequence of the protein.As is well known in the art, proteins transported across the cellmembrane typically have an N-terminal sequence rich in hydrophobic aminoacids about 15 to 30 amino acids long. Sometime during the process ofpassing through the membrane, the signal sequence is cleaved by signalpeptidase. Watson et al., Molecular Biology of the Gene, p. 731, 1987.Thus, the signal peptide encoding sequence of the tomatoendo-1,4-β-glucanase gene may be linked to another, foreign, structuralgene to provide for transport of the foreign gene product to the cellwall. The foreign structural gene may be derived from any sourceincluding bacteria, yeast, animals or plants. Typically, the signalpeptide encoding sequence will be joined at its 3' end to a linker forattachment to the foreign structural gene in the proper reading frame.Foreign genes of interest include carbohydrate and cell wallmetabolizing enzymes, such as invertase, dextransucrase, levansucrase.Also of interest are genes that encode proteins involved in diseaseresistance such as chitinase, hydroxyprotein-rich glycoproteins, andpolygalacturonase inhibiting proteins.

III. Vector construction

The desired recombinant vector will comprise an expression cassettedesigned for initiating transcription of the antisense cDNA in plants.Companion sequences, of bacterial or viral origin, are also included toallow the vector to be cloned in a bacterial or phage host.

The vector will preferably contain a broad host range prokaryote originof replication. A selectable marker should also be included to allowselection of bacterial cells bearing the desired construct. Suitableprokaryotic selectable markers include resistance to antibiotics such askanamycin or tetracycline.

Other DNA sequences encoding additional functions may also be present inthe vector, as is known in the art. For instance, in the case ofAgrobacterium transformations, T-DNA sequences will also be included forsubsequent transfer to plant chromosomes.

A bacterial expression vector may be used if expression of the glucanasecDNA in bacteria is desired. Insertion of an expression vector intobacteria is useful in biomass conversion of plant tissues to ethanol ormethanol. Construction of a bacterial expression vector is typicallydone by placing the cDNA downstream from a strong bacterial promoter.Examples of bacterial promoters that might be used include β-lactamase,β-galactosidase, and the phage λpL promoters. The efficiency oftranslation of mRNA in bacteria is critically dependent on the presenceof a ribosome-binding site and its distance from the transcriptioninitiation codon.

For expression in plants, the recombinant expression cassette willcontain in addition to the desired sequence, a plant promoter region, atranscription initiation site (if the sequence to be transcribed lacksone), and a transcription termination sequence. Unique restrictionenzyme sites at the 5' and 3' ends of the cassette are typicallyincluded to allow for easy insertion into a pre-existing vector.##STR2##

Sequences controlling eukaryotic gene expression have been extensivelystudied. Promoter sequence elements include the TATA box consensussequence (TATAAT), which is usually 20 to 30 base pairs (bp) upstream ofthe transcription start site. In most instances the TATA box is requiredfor accurate transcription initiation. By convention, the start site iscalled +1. Sequences extending in the 5' (upstream) direction are givennegative numbers and sequences extending in the 3' (downstream)direction are given positive numbers.

In plants, further upstream from the TATA box, at positions -80 to -100,there is typically a promoter element with a series of adeninessurrounding the trinucleotide G (or T) N G. J. Messing et al., inGenetic Engineering in Plants, pp. 221-227 (Kosage, Meredith andHollaender, eds. 1983). Other sequences conferring tissue specificity,response to environmental signals, or maximum efficiency oftranscription may also be found in the promoter region. Such sequencesare often found within 400 bp of transcription initiation size, but mayextend as far as 2000 bp or more.

In the construction of heterologous promoter/structural genecombinations, the promoter is preferably positioned about the samedistance from the heterologous transcription start site as it is fromthe transcription start site in its natural setting. As is known in theart, however, some variation in this distance can be accommodatedwithout loss of promoter function.

The particular promoter used in the expression cassette is a noncriticalaspect of the invention. Any of a number of promoters which directtranscription in plant cells is suitable. The promoter can be eitherconstitutive or inducible. Promoters of bacterial origin include theoctopine synthase promoter, the nopaline synthase promoter and otherpromoters derived from native Ti plasmids. Herrara-Estrella et al.,Nature, 303:209-213, 1983. Viral promoters include the 35S and 19S RNApromoters of cauliflower mosaic virus. Odell et al. Nature, 313:810-812,1985. Possible plant promoters include the ribulose-1,3-bisphosphatecarboxylase small subunit promoter and the phaseolin promoter. Thepromoter sequence from the E8 gene and other genes in which expressionis induced by ethylene may also be used. The isolation and sequence ofthe E8 promoter is described in detail in Deikman and Fischer, EMBO J.7:3315-3327, 1988. which is incorporated herein by reference.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

If the mRNA encoded by the structural gene is to be efficientlytranslated, polyadenylation sequences are also commonly added to thevector construct. Alber and Kawasaki, Mol. and Appl. Genet, 1:419-434,1982. Polyadenylation is of importance for expression of the glucanasecDNA in plant cells. Polyadenylation sequences include, but are notlimited to the Agrobacterium octopine synthase signal (Gielen et al.,EMBO J., 3:835-846, 1984) or the nopaline synthase signal (Depicker etal., Mol. and Appl. Genet, 1:561-573, 1982).

The vector will also typically contain a selectable marker gene by whichtransformed plant cells can be identified in culture. Usually, themarker gene will encode antibiotic resistance. These markers includeresistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin.After transforming the plant cells, those cells having the vector willbe identified by their ability to grow an a medium containing theparticular antibiotic.

IV. Transcription of endo-1,4-β-glucanase antisense cDNA in Plant CellsA. Transformation of Plant Cells by In Vitro Techniques 1. DirectTransformation

The vector described above can be microinjected directly into plantcells by use of micropipettes to mechanically transfer the recombinantDNA. Crossway, Mol. Gen. Genetics, 202:179-185, 1985. The geneticmaterial may also be transferred into the plant cell using polyethyleneglycol, Krens, et al., Nature, 296, 72-74, 1982.

Another method of introduction of nucleic acid segments is high velocityballistic penetration by small particles with the nucleic acid eitherwithin the matrix of small beads or particles, or on the surface, Klein,et al., Nature, 327, 70-73, 1987.

Yet another method of introduction is fusion of protoplasts with otherentities, either minicells, cells, lysosomes or other fusiblelipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA. 79,1859-1863, 1982.

The DNA may also be introduced into the plant cells by electroporation.Fromm et al., Pro. Natl Acad. Sci. USA, 82:5824 (1985). In thistechnique, plant protoplasts are electroporated in the presence ofplasmids containing the expression cassette. Electrical impulses of highfield strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and regenerate.

2. Vectored Transformation

Cauliflower mosaic virus (CaMV) may be used as a vector for introducingthe antisense DNA into plant cells. (Hohn et al., 1982 "MolecularBiology of Plant Tumors." Academic Press, New York, pp. 549-560; Howell,U.S. Pat. No. 4,407,956). In accordance with the described method, theentire CaMV viral DNA genome is inserted into a parent bacterial plasmidcreating a recombinant DNA molecule which can be propagated in bacteria.After cloning, the recombinant plasmid is further modified byintroduction of the desired sequence into unique restriction sites inthe viral portion of the plasmid. The modified viral portion of therecombinant plasmid is then excised from the parent bacterial plasmid,and used to inoculate the plant cells or plants.

Another method of introducing the DNA into plant cells is to infect aplant cell with Agrobacterium tumefaciens or A. rhizogenes previouslytransformed with the gene. Under appropriate conditions known in theart, the transformed plant cells are grown to form shoots or roots, anddevelop further into plants.

Agrobacterium is a representative genus of the gram-negative familyRhizobiaceae. Its species are responsible for crown gall (A.tumefaciens) and hairy root disease (A. rhizogenes). The plant cells incrown gall tumors and hairy roots are induced to produce amino acidderivatives known as opines, which are catabolized only by the bacteria.The bacterial genes responsible for expression of opines are aconvenient source of control elements for chimeric expression cassettes.In addition, assaying for the presence of opines can be used to identifytransformed tissue.

Heterologous genetic sequences can be introduced into appropriate plantcells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid ofA. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells oninfection by Agrobacterium and is stably integrated into the plantgenome. J. Schell, Science, 237: 1176-1183, 1987.

Ti and Ri plasmids contain two regions essential for the production oftransformed cells. One of these, named transferred DNA (T-DNA), istransferred to plant nuclei and induces tumor or root formation. Theother, termed the virulence (vir) region, is essential for the transferof the T-DNA but is not itself transferred. The T-DNA will betransferred into a plant cell even if the vir region is on a differentplasmid. Hoekema, et al., Nature, 303:179-189, 1983. The transferred DNAregion, can be increased in size by the insertion of heterologous DNAwithout its ability to be transferred being affected. A modified Ti orRi plasmid, in which the disease-causing genes have been deleted, can beused as a vector for the transfer of the gene constructs of thisinvention into an appropriate plant cell.

Construction of recombinant Ti and Ri plasmids in general followsmethods typically used with the more common bacterial vectors, such aspBR322. Additional use can be made of accessory genetic elementssometimes found with the native plasmids and sometimes constructed fromforeign sequences. These may include but are not limited to "shuttlevectors", (Ruvkun and Ausubel, 1981, Nature 298:85-88), promoters,Lawton et al., 1987, Plant Mol. Biol. 9:315-324) and structural genesfor antibiotic resistance as a selection factor (Fraley et al., Proc.Nat. Acad. Sci., 80:4803-4807, 1983).

All plant cells which can be transformed by Agrobacterium and from whichwhole plants can be regenerated can be transformed according to thepresent invention to produce transformed intact plants which contain thedesired DNA. There are two common ways to transform plant cells withAgrobacterium:

(1) co-cultivation of Agrobacterium with cultured isolated protoplasts,or

(2) transformation of intact cells or tissues with Agrobacterium.

Method (1) requires an established culture system that allows forculturing protoplasts and subsequent plant regeneration from culturedprotoplasts.

Method (2) requires (a) that the intact plant tissues, such ascotyledons, can be transformed by Agrobacterium and (b) that thetransformed cells or tissues can be induced to regenerate into wholeplants.

Most dicot species can be transformed by Agrobacterium. All specieswhich are a natural plant host for Agrobacterium are transformable invitro. Monocotyledonous plants, and in particular, cereals, are notnatural hosts to Agrobacterium. Attempts to transform them usingAgrobacterium have been unsuccessful until recently. Hooykas-VanSlogteren et al., Nature, 311:763-764, 1984. There is growing evidencenow that certain monocots can be transformed by Agrobacterium. Usingnovel experimental approaches cereal species such as rye (de la Pena etal., Nature 325:274-276, 1987), corn (Rhodes et al., Science240:204-207, 1988), and rice (Shimamoto et al., Nature 338:274-276,1989) may now be transformed.

B. Selection and Regeneration of Transformed Plant Cells

After transformation, transformed plant cells or plants comprising theantisense DNA must be identified. A selectable marker, such as thosediscussed, supra, is typically used. Transformed plant cells can beselected by growing the cells on growth medium containing theappropriate antibiotic. The presence of opines can also be used if theplants are transformed with Agrobacterium.

After selecting the transformed cells, one can confirm expression of thedesired heterologous gene. Simple detection of mRNA encoded by theinserted DNA can be achieved by well known methods in the art, such asNorthern blot hybridization. The inserted sequence can be identified bySouthern blot hybridization, as well. See, e.g., Sambrook, supra.

After determination of the presence of the antisense DNA, whole plantregeneration is desired. All plants from which protoplasts can beisolated and cultured to give whole regenerated plants can betransformed by the present invention. Some suitable plants include, forexample, species from the genera Fragaria, Lotus, Medicago, Onobrychis,Trifolium, Trigonella. Vigna, Citrus, Linum, Geranium, Manihot, Daucus.Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis,Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium,Zea, Triticum, Sorghum, Malus, Apium, and Datura.

Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co.New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III,1986.

It is known that practically all plants can be regenerated from culturedcells or tissues, including but not limited to, all major species ofsugarcane, sugar beet, cotton, fruit trees, and legumes.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining transformed explants is first provided. Callus tissue isformed and shoots may be induced from callus and subsequently rooted.Alternatively, embryo formation can be induced in the callus tissue.These embryos germinate as natural embryos to form plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins. It is also advantageous to add glutamic acid andproline to the medium, especially for such species as corn and alfalfa.Efficient regeneration will depend on the medium, on the genotype, andon the history of the culture. If these three variables are controlled,then regeneration is usually reproducible and repeatable.

After the expression cassette is stably incorporated in transgenicplants, it can be transferred to other plants by sexual crossing. Any ofa number of standard breeding techniques can be used, depending upon thespecies to be crossed.

V. Definitions

The phrase "DNA sequence" refers to a single or double-stranded polymerof deoxyribonucleotide bases read from the 5' to the 3' end. It includesboth self-replicating plasmids, infectious polymers of DNA andnon-functional DNA.

The term "promoter" refers to a region of DNA upstream from thestructural gene and involved in recognition and binding RNA polymeraseand other proteins to initiate transcription. A "plant promoter" is apromoter capable of initiating transcription in plant cells.

The term "plant" includes whole plants, plant organs (e.g., leaves,stems, roots, etc.), seeds and plant cells.

The phrase "suitable host" refers to a microorganism or cell that iscompatible with a recombinant plasmid, DNA sequence or recombinantexpression cassette and will permit the plasmid to replicate, to beincorporated into its genome, or to be expressed.

The term "expression" refers to the transcription and translation of astructural gene so that a protein is synthesized.

A "constitutive" promoter is a promoter which is active under allenvironmental conditions and all states of development or celldifferentiation.

An "inducible" promoter is a promoter which is under environmental ordevelopmental control. Examples of environmental conditions that mayeffect transcription by inducible promoters include anaerobic conditionsor the presence of light. Examples of promoters under developmentalcontrol include promoters that initiate transcription only in certaintissues, such as the promoter from the E8 gene which is induced byethylene in ripening fruit.

The term "opposite orientation for expression" refers to adouble-stranded DNA sequence from a structural gene that is inserted inan expression cassette in an inverted manner with respect to itsnaturally occurring orientation. Specifically, the strand that isnormally the "template strand becomes the coding strand, and vice versa.

The term "uninterrupted" refers to a DNA sequence containing an openreading frame that lacks intervening, untranslated sequences.

The term "non-wild type DNA" refers to DNA sequences that do not flank agiven DNA sequence in its naturally occurring environment.

The following experimental results are offered by way of example and notby way of limitation.

EXAMPLES A. Preparation of Tomato endo-1,4-β-glucanase cDNA 1. cDNALibrary Production

A vector-primed cDNA library was prepared using standard methods. Thelibrary was prepared in the cloning vector pARC7 from ripe tomato fruitpoly-A RNA by the method of Alexander et al., Gene, 31:79-89, 1984,which is incorporated herein by reference.

2. cDNA Library Screening a. Growing Colonies

HB101 cells containing a red ripe tomato-derived cDNA library weretitered and dilutions were made to give approximately 5000 colonies per10 ml of Luria Broth (LB). Ten ml aliquots of chilled bacterialsuspension were vacuum filtered onto ten 132 mm nitrocellulose filters,which were then placed colony sides up on LB-agar plates containing 100μg/ml ampicillin. Plates were incubated at 37° C. until colonies wereapproximately 0.5 mm in diameter.

b. Replica Plating

Master filters were removed from plates, numbered and given orientationmarks with black ink. A fresh filter was wetted on a fresh LB plate andwas laid on top of each master filter and orientation marks copied tothe replicate. This process of colony transfer was repeated with a 2ndfresh filter to give two replica filters per master filter. Replicateswere grown on LB-agar plates at 37° C. until colonies were approximately0.5 mm and then were transferred to plates containing LB-agar with 150μg/ml chloramphenicol. These were grown 12 hours at 37° C.

c. Bacterial Colony Lysis

Replica filters were removed from plates and placed colony sides up atroom temperature on sheets of Whatman 3MM paper wetted with 0.5MNaOH/1.5M NaCl. After 10 minutes, filters were blotted on dry 3MM paperand transferred for 2 minutes to 3MM paper wetted with 1M Tris pH 7/1.5MNaCl. Filters were immersed in 3×SSC for 15 seconds, placed on dry 3MMpaper and air dried prior to baking at 80° C. under vacuum for 2 hours.

d. Hybridization to Oligonucleotide Probe

Bacterial debris was removed from baked filters by washing with3×SSC/0.1% SDS at 62° C. for 24 hours, during which time wash solutionwas replaced with fresh solution 3 times. Filters were collectivelyprehybridized at 37° C. overnight with 6×SSC, 1×Denhardts Solution, 0.5%SDS, 0.05% NaPPi and 0.1 mg/ml boiled and ice-quenched salmon sperm DNA.The 20 filters were then divided into two groups of replicates forhybridization.

Two 26 base oligonucleotide probe were synthesized at a DNA synthesizingfacility. Probe sequences corresponded to two regions of glucanase thatare completely conserved at the amino acid level in bean abscission zoneglucanase and avocado fruit glucanase. Oligonucleotides were solubilizedin 10 mM Tris-EDTA (TE) pH 8 and extracted with TE-saturated butanol;they were then adjusted to 0.3M in ammonium acetate and wereprecipitated with 4 volumes of ethanol at -80° C. DNA was harvested bycentrifugation and was brought to 1 mg/ml in TE pH 8.

One μg of each oligonucleotide probe was end labeled with 32P-ATPaccording to the T4 DNA Polymerase Labeling System (Bethesda ResearchLabs) protocol supplied by the manufacturer. Specific activity of eachprobe exceeded 5×10⁷ cpm/μg.

Each set of replica filters was incubated overnight at 42° C. in ahybridization bag containing 15 ml of hybridization buffer and one ofthe boiled and ice-quenched radiolabeled probes. Hybridization mediumwas 6×SSC, 1×Denhardt's solution, 0.05% NaPPi and 0.1 mg/ml boiled andice-quenched salmon sperm DNA.

Filters were washed at 42° C. in 6×SSC, 0.05% NaPPi for several hourswith several buffer changes. They were then exposed to Kodak X-O-Mat ARfilm at -80° C. for 24 hours using an intensifying screen. Film wasdeveloped and clones containing glucanase probe sequence were identifiedvia the comparison of orientation marks on the film with those on thecorresponding master plate.

e. Secondary Screening of Putative Glucanase Clones

Colonies identified by the glucanase oligonucleotide probes were pickedwith sterile toothpicks, dispersed into 1 ml LB and incubated withshaking at 37° C. for 2.5 hours. Suspensions were then diluted500,000-fold and vacuum filtered in 5 ml aliquots of chilled LB through82 mm nitrocellulose filters. These were grown at 37° C. on LB agar with100 μg/ml ampicillin for 8 hours prior to their transfer to LB agarplates containing 150 μg/ml chloramphenicol. These were then incubatedat 37° C. for 12 hours. Filters were processed and screened withradiolabeled oligonucleotides probes as per steps 3 and 4 above. Singlecolonies of each of the 28 glucanase clones identified in the secondaryscreen were picked into 3 ml of LB ampicillin and grown overnight at 37°C.

f. Southern Analysis of Glucanase Clones

Mini prep DNA was isolated from bacterial cultures by the method ofKraft et al. Biotechnicues 6(6):544-546 which is incorporated herein byreference. DNA was then digested with Sma I restriction enzyme for 2.5hours under standard conditions to release the cloned glucanase insertsfrom their respective pArc vectors; digestion products were sizefractionated on 1.2% agarose gels using avocado glucanase cDNA andtomato polygalacturonase cDNA clones as positive and negative controls,respectively. Following incubation in 250 mM HCl, followed by 0.5MNaOH/1.5M NaCl and finally by 0.5M Tris/3M NaCl, gels were blotted tonitrocellulose and probed with each oligonucleotide probe end labeled aspreviously described. The largest glucanase insert was estimated to be1.8 kilobases, similar to the previously characterized 1.9 kB avocadoglucanase cDNA. This clone was selected for sequencing.

3. Sequencing of Tomato Glucanase a. Subcloning of Tomato Glucanase

Sma I digestion of mini prep DNA prepared from the colony described instep 6 of part 1 released the 1.8 kB (estimated size) glucanase clonefrom the pArc vector. Digestion products were precipitated with 0.4volumes ammonium acetate and 2 volumes ethanol and resuspended in 1×DNAsample buffer. Products were loaded onto a low-melt agarose gel withinsert separated from vector by electrophoresis at 80 V. The insert wasexcised from the gel and stored as a gel slice at -20° C. untilrequired. DNA concentration was estimated from the relative intensitiesof ethidium bromide staining between insert and defined standards.

Bluescript vector (SK+) (Stratagene Inc., La Jolla, Calif.) waslinearized by Sma I digestion under standard conditions at 30° C. After2.5 hours, digested vector was extracted once withphenol:chloroform:isoamyl alcohol (25:24:1) and once withchloroform-isoamyl alcohol (24:1) prior to precipitation with 0.4volumes ammonium acetate and 2.5 volumes ethanol. The pelleted DNA wasbrought up in 500 μl 50 mM Tris, 0.1 mM EDTA, pH 8 and wasdephosphorylated with Boehringer Mannheim calf intestine alkalinephosphatase as per the manufacturer's instructions for blunt ended DNAfragments. Dephosphorylated vector was harvested by ammoniumacetate/EtOH precipitation as described previously and was brought to100 μg/ml with water.

Dephosphorylated vector was ligated at 15° C. for 12 hours to meltedglucanase insert from the low melt agarose gel. Ligation specificationswere as follows for each 45 μl ligation: total DNA concentration=1 μg,insert:vector=2.1 on a molar basis. T4 DNA ligase=100 units/ml, finalPEG concentration=5%.

Ligation mixtures were brought up to 100 μl with TE 8.0 and added to 200μl freshly thawed XL1 Blue competent cells. After 30 minutes on ice,cells were heat shocked 5 minutes at 42° C. and added to 4 ml 2XL mediumwhich had been prewarmed to 37° C. Cells were shaken at 100 rpm on anorbital shaker for 100 minutes at 37° C. and transferred to ice.Appropriate aliquots of the cells were then spread on LB agar platescontaining 100 μg/ml ampicillin and 50 μg/ml tetracycline. Plates hadbeen pre-spread with 100 μl of (50 μl 100 mM IPTG, 20 μl 20 mg/ml X-gal,30 μl LB). Plates were then incubated overnight at 37° C., at which timetransformed colonies (white) could be distinguished from non-transformedcolonies (blue). Miniprep DNA was isolated from transformants aspreviously described and digested with Sma I to release inserts. Oneglucanase transformant of approximately 1.8 kB was identified followingthe electrophoretic separation of digestion products on a 1.5% agarosegel. Double stranded miniprep DNA was prepared as previously describedfor sequencing purposes.

b. Sequencing

Double stranded DNA templates of varying lengths for use in first strandsequencing were generated by exonuclease digestion of glucanase miniprepDNA as described in the Erase-A-Base kit (Promega) protocol supplied bythe manufacturer. Sequencing was conducted by the dideoxy method(Sanger, et al., Proc. Nat. Acad. Sci. USA, 74:5463-5467) outlined fullyin the Sequenase kit (United States Biochemical Co.) protocol providedby the manufacturer. Reverse M13 primer was purchased from Pharmacia.

Sequence data generated was entered and analyzed using the Microgeniesequence analysis computer program (Beckman Instruments, Inc.) strandresulted from the overlap of over 20 smaller sequences.

B. Vector Construction

Four different vectors were constructed. One vector, E8antiCL, containsthe promoter from the tomato E8 gene and glucanase antisense DNA. Thispromoter is inducible in ripening tomato fruit. The second vector,CaMVantiCL, contains the cauliflower mosaic virus 35S promoter andglucanase antisense DNA. This promoter is constitutive. The other twovectors were constructed in the same manner but with the addition ofpolygalacturonase antisense DNA and appropriate promoters. Theconstruction of the latter two vectors is illustrated in FIG. 2

1. E8antiCL

A 2.0 kb E8 promoter fragment was isolated by cleaving pE8mutRN2.0 withNcol. The preparation of pE8mutRN2.0 is described in Giovaninnoni etal., The Plant Cell, 1:53-63, 1989, which is incorporated herein byreference. The 5' overhang of the Ncol restriction site was blunt-endedwith the large fragment of DNA polymerase (Klenow fragment) and digestedwith EcoR1 restriction endonuclease. The resulting 2.0 kb EcoR1/filledNcol fragment was ligated into pUC118 cleaved with EcoR1 and Sma1restriction endonucleases. The resulting construction, pE8mutRN2.0(+),retains the original Ncol restriction site and includes BamH1, Xbal,Sal1, Pst1, Sph1, and Hindlll sites downstream of the Ncol restrictionsite.

The 1.8 kb endo-1,4-β-glucanase cDNA cloned into the Sma1 site of theBluescript M13+ (SK+) vector (Stratagene Inc., La Jolla, Calif.) wasliberated by digestion with BamH1 and Kpn1 followed by agarose gelpurification. The fragment was then ligated into BamH1/Kpn1 digestedpUC118 to generate pUCCL1.8. The 1.8 kb BamH1/Sst1 cDNA insert ofPUCCL1.8 was liberated by restriction endonuclease digestion andpurified by agarose gel electrophoresis.

The resulting 1.8 kb BamH1/Sst1 fragment was utilized in a tri-molecularligation with the 0.25 kb Sst1/EcoR1 Agrobacterium nopaline synthasegene transcription terminator fragment (capable of directing terminationof gene transcription in plants) purified from pBl121 (Clonetech Inc.,Palo Alto, Calif.) and ligated into pUC118 cleaved with BamHI and EcoRI.The resulting pUCantiCL-ter construction contained the glucanase cDNAfused at its 5' end to the nopaline synthase gene transcriptiontermination tragment via Sst1 site ligation. The 2.05 kb antiCL-terfragment was isolated from pUCantiCL-ter by digestion with BamH1followed by partial digestion with EcoR1. The 2.05 kb product was thenpurified on a agarose gel.

The resulting 2.05 kb EcoR1/BamH1 fragment was utilized in atri-molecular ligation with the 2.0 kb EcoR1/BamH1 fragment purifiedfrom pE8mutRN2.0 and pUC118 cleaved with EcoR1. The resultingconstruction, pE8antiCL, contains the E8 promoter fused to the 3' end ofthe glucanase cDNA clone with the 5' end fused to the transcriptiontermination fragment of the nopaline synthase gene. The internal EcoR1site located between the cDNA and transcription terminator sequences wasremoved by partial digestion with EcoR1 restriction endonucleasefollowed by filling in of the EcoR1 5' overhang with Klenow enzyme andsubsequent ligation of the filled in EcoR1 restriction endonucleasesites. The loss of the internal EcoR1 site was verified by restrictionendonuclease mapping of the resulting construction, pE8antiCL-R1. The4.05 kb insert of pE8antiCL-R1 was liberated with EcoR1 restrictionendonuclease, purified by agarose gel electrophoresis, and ligated intothe EcoR1 site of the Agrobacterium T-DNA cointegrative shuttle vectorpMLJ1, described in subsection 3, infra. The resulting construction isdesignated pMLJ1:E8antiCL.

2. CaMVantiCL

Regulatory sequences of the Cauliflower Mosaic Virus 35s transcriptionunit Were isolated from pBI121 (Clonetech Inc., La Jolla, Calif.) bydigestion with SphI and BamHI followed by agarose gel purification. Theresulting 0.8 kb SphI/BamHI fragment was employed in a tri-molecularligation with the 2.05 kb BamHI/EcoRI fragment of pUCantiCL-ter(described above) and pUC118 digested with Sph1 and EcoR1. The resultingconstruction was partially digested with EcoR1, and subjected to afill-in reaction with Klenow enzyme followed by ligation to remove theinternal EcoR1 restriction endonuclease site located between the 5' endof the cDNA and the plant transcription termination sequences.Restriction endonuclease mapping was employed to verify that the EcoR1site between the cDNA and transcription termination sequences wasremoved. The resulting construction was designated pCaMVantiCL-S.pCaMVantiCL-S was digested with Sph1. The 3' overhang resulting fromSph1 digestion was filled in using T4 DNA polymerase and ligated toEcoRI linkers (BRL, Bethesda, Md.). The resulting construction wastermed pCaMVantiCL. The 2.85 kb insert of pCaMVantiCL was isolated viadigestion with EcoR1 restriction endonuclease followed by agarose gelpurification and ligated into the EcoRI site of pMLJ1 to generate pMLJ1:CaMVantiCL.

3. E8antiPG/CL

The 1.7 kb full length tomato fruit polygalacturonase cDNA insert ofpBSPG1.9 (DellaPenna et al., Plant Physiology 90:1372-1377, 1989 whichis incorporated herein by reference), cloned into the SmaI site of theBluescript M13+ (SK+) vector was liberated by digestion with Sal1 andSst1 restriction endonucleases followed by agarose gel purification. Theresulting 1.7 kb fragment was utilized in a tri-molecular ligation withthe 0.25 kb Sst1/EcoR1 Agrobacterium nopaline synthase genetranscription termination sequence (described above) and Sal1/EcoR1digested pUC118. The resulting construction was designated pUCantiPG-terand consists of the 5' end of the polygalacturonase cDNA clone fused tothe nopaline synthase transcription termination sequence at the Sst1site.

The 1.95 kb insert of pUCantiPG-ter was liberated by digestion with Sal1and EcoR1 restriction endonucleases followed by agarose gelpurification. The resulting 1.95 kb Sal1/EcoR1 fragment was utilized ina tri-molecular ligation with the 2.0 kb EcoR1/Sal1 E8 promoter fragmentisolated from pE8mutRN2.0(+) (described above) and pUCE8antiPG.

The 3.95 kb insert of pUCE8antiPG was isolated by agarose gelpurification following digestion with EcoR1 restriction endonuclease andsubsequent DNA polymerase (Klenow) fill-in of the 5' EcoR1 overhangsbordering both sides of the 3.95 kb antisense gene. The unique Sal1restriction site of the cointegrative plant transformation vector,pMLJ1, was cleaved with Sal1 and filled in with Klenow enzyme. The bluntended 3.95 kb E8antiPG fragment was ligated into the blunt ended Sal1site of pMLJ1 to form pMLJ1:E8antiPG. pMLJ1:E8antiPG was cleaved in theunique EcoR1 site of the pMLJ1 sequences. The 4.05 kb insert ofpE8antiCL-R1 (described above) was liberated with EcoR1 and purified byagarose gel electrophoresis. The resulting 4.05 kb E8antiCL-R1 fragmentwas ligated into the EcoR1 site of pMLJ1:E8antiPG to formpMLJ1:E8antiPG/CL (see FIG. 2).

4. CaMVantiPG/CL

Regulatory sequences of the Cauliflower Mosaic Virus 35S transcriptionunit were isolated from pBI121 as described above. The 1.95 kb insert ofpUCantiPG-ter (described above) was isolated by digestion with EcoRI andpartial digestion with BamHI, followed by agarose gel purification ofthe resulting 1.95 kb fragment. The resulting 0.8 kb Sph1/BamH1 fragmentof the CaMV 35S promoter was employed in a tri-molecular ligation withthe 1.95 kb BamHI/EcoR1 insert of pUCantiPG-ter and pUCI18 digested withSph1 and EcoR1 restriction endonucleases to produce the constructiondesignated pUCCaMVantiPG-S co. pUCCaMVantiPG-S was digested with Sph1.The 3' overhang resulting from Sph1 digestion was filled in using T4 DNApolymerase and ligated to EcoRI linkers (BRL, Bethesda, Md.). Theresulting construction was termed pUCCaMVantiPG and contains the 2.75 kbCaMVantiPG gene cloned into the EcoR1 site of pUC118.

The 2.85 kb insert of pCaMVantiCL was isolated by agarose gelelectrophoresis following digestion with EcoR1 restriction endonucleaseand filling in of the 5' EcoR1 overhangs with Klenow enzyme. The uniqueSal1 site of pMLJ1 was cleaved with Sal1 and filled in with Klenowenzyme. The 2.85 kb blunt end CaMVantiCL fragment was ligated into theEcoR1 site of pMLJ1:CaMVantiCL2 to form pMLJ1:CaMVantiPG/CL (see FIG.2).

5. Co-integration of Antisense Gene Constructions

Triparental mating was done according to methods well known in the artas described in Van Haute et al., EMBO J. 411-417, 1983, which isincorporated herein by reference. The shuttle vector used in thetriparental mating is not a critical aspect of the invention. Theparticular shuttle vector used here, pMLJ1, is derived from thatdescribed in DeBloch et al., EMBO J. 3:1681-1689, 1984.

Triparental mating of E. coli (strain JM109) harboring pMLJ1:E8antiCL,pMLJ1:CaMVantiCL, pMLJ1:E8antiPG/CL, or pMLJ1:CamVantiPG/CL withAgrobacterium tumefaciens containing the cointegrative planttransformation vector pGV3850 (this vector is described in detail inZambryski et al., EMBO J. 2:2143, 1983, which is incorporated herein byreference) and the helper E. coli strain pGJ23 resulted in cointegrationof the antisense gene constructions into pGV3850, pGV3850:E8antiCL andpGV3850:CaMVantiCL were utilized to insert antisenseendo-1,4-β-glucanase sequences into the tomato genome.

C. Transformation of Tomato With Antisense Endo-1,4-β-GlucanaseConstructions Summary of the Procedure

In brief, sterile cotyledon pieces were infected with Agrobacteriumcontaining a Ti plasmid which includes within the T-DNA a neomycinphosphotransferase gene (NPT11) capable of conferring kanamycinresistance in transgenic plants. The co-integrative Agrobacteriumtumefaciens Ti vector, pGV3850, with pMLJ1:E8antiCL, pMLJ1:CaMVantiCL,pMLJ1:E8antiPG/CL or pMLJ1:CamVantiPG/CL independently integrated intoit, was used to transfer the two antisense gene constructions intoindependent tomato genomes. Co-cultivation of tomato (Lycopersiconesculentum cv Ailsa Craig) cotyledon pieces with the bacteria took placefor 48 hours on tobacco feeder plates. The feeder cells increase theefficiency of transformation of tomato after the co-cultivation process.Regeneration of shoots was induced on the regeneration medium. From thisstage on, antibiotics were used to inhibit the growth of Agrobacterium(Cefotaxime) and to select for transformed plant cells (kanamycin).Finally, shoots were transferred to rooting medium and then planted insoil and grown in the greenhouse.

1. Maintenance of Feeder Cells

To maintain the tobacco Xanthi suspension culture the cells werefiltered through a 40 mesh filter once per week. 10 mls of filtrate wereadded to 100 mls of fresh Xanthi medium in a 500 ml flask.

2. Tomato Seed Germination

Approximately 50 seeds in a 50 ml beaker were stirred in 20 mls 70% EtOHfor 2 minutes and rinsed with sterile water. They were then stirred 5minutes in 20% bleach with 2 drops of Tween 80 and rinsed 4 times withsterile distilled H₂ O.

Using sterile forceps, 12 to 15 seeds were placed on each plate. Thepetri plate was wrapper with parafilm and aluminum foil and grown at 25°C. After 5 days (when the seeds had reached about 60% germination), theywere removed from the aluminum foil and grown under 2500 lux, with a 16hour photoperiod. The seedlings were grown for a total of 8 days.

3. Preparation of Feeder Plates

Thick petri plates of approximately 40 mls of Xanthi suspension culturemedium with 8 g/l agar were employed. 1 ml of a thick Xanthi suspensionculture (7 days old) was pipetted onto each feeder plate. The plateswere sealed with parafilm and incubated for 12 hours in the growthchamber (25° C.) on a lighted shelf.

4. Putting Cotyledons on the Feeder Plates

A sterile Whatman #1 filter was placed onto each feeder cell plate.Cotyledons were cut with a scalpel in a drop of sterile water in a petriplate. The scalpel was rocked gently to make the cuts thus minimizingtearing and bruising of the tissue. Only the ends of the cotyledons werecut off. Cut cotyledons were placed onto the filter paper on the feederplate upside-down (cuticle side down). Approximately 50 cotyledon pieceswere placed on each plate. The plates were sealed with parafilm andplaced in the growth chamber for 16 hours.

5. Infection with Transformed Agrobacterium

10 ml overnight cultures of the Agrobacterium containing pMLJ1:E8antiCLand pMLJ1:CaMVantiCL were grown in YE8 medium supplemented with 25 μg/mlspectinomycin. Agrobacterium overnight cultures were diluted four-foldin the seed germination medium to an O.D. of 590. 0.5 mls of dilutedbacteria were aliquoted into a petri dish followed by addition 30cotyledon pieces previously co-cultivated with tobacco feeder cells. TheAgrobacterium/cotyledon mixture was swirled to wet. The cotyledons werewet in the bacteria for 5 minutes. The cotyledons were touched once to asterile paper towel. Cotyledons were placed back on the same feederplates upside-down and co-cultivated for an additional 48 hours.

6. Regeneration

After co-cultivation with the bacteria, cotyledons were placed on theregeneration medium right-side-up. The edges of the cotyledon will curldown into the agar insuring the wounded surfaces will be in directcontact with the drugs. 15 cotyledon pieces were placed on each plate.

Within 10 days callus was visible at the edges of the infectedcotyledons. Cotyledon pieces were transferred to fresh plates every 2weeks. Shoots and dark green callus was transferred to shooting medium(same as regeneration medium except that the zeatin concentration isreduced to 0.1 mg/ml). After 6 weeks (3 transfers) all callus and shootswere transferred to shooting medium.

For rooting, TM5 rooting medium was employed. (Shahin, Theor. Appl. Gen.69: 235-240, 1985). The levels of kanamycin and cefatoxime are reducedto 25 mg/l and 125 mg/l, respectively.

After the shoots developed sufficient roots, they were transferred tosoil. To transfer plants to soil, they were gently removed from the agarusing a spatula to scrape away most of the agar. The roots were rinsedin warm water to remove as much agar as possible. They were planted inclay pots which were placed inside GA-7 boxes. The covers of the boxeswere gradually opened over several days and watered with 1/2-strengthHoagland's solution every other watering. After 2 weeks, the plants werecompletely uncovered in the growth chamber and were transplanted intolarge pots and moved to the greenhouse.

    ______________________________________                                        7. Media                                                                      a. Xanthi Suspension Culture Medium                                                                        stock                                            ______________________________________                                        1 bottle KC MS Salts (MM100)                                                                       4.3 g                                                    i-inositol          100 mg                                                    sucrose              30 g                                                     KH.sub.2 PO4         2 mls   100 mg/ml                                        thiamine             1.3 mls  1 mg/ml                                         2,4-D                2 mls   100 mg/l                                         kinetin              0.4 mls  0.25 mg/ml                                      pH 5.5 with KOH                                                               H.sub.2 O to 1 liter                                                          aliquot 100 mls into 500 ml flasks                                            plug the flasks and cap with aluminum foil                                    autoclave 20'                                                                 b. Plates for seed germination                                                MS Medium          1 pkg. KC MM-100                                           3% sucrose         30 g sucrose                                                                  800 mls H.sub.2 O                                          pH to 5.7 with KOH                                                            volume to 1 liter                                                             add 8 g bacto agar (0.8% agar)                                                autoclaved 20 minutes                                                         poured into thick petri plates (about 30 mls per                              plate)                                                                        c. Regeneration medium                                                        for 1 liter:                                                                  4.3 g MS Salts (KC MM-100)                                                    30 g glucose                                                                  0.59 g MES                                                                    2 ml 500X Gamborgs vitamins (see below)                                       ph to 5.8 with 1N KOH                                                         volume to 1 liter                                                             8 g tissue culture grade agar                                                 Autoclave 20 minutes                                                          Cool to 50 degrees C.                                                         Add:                                                                          1 mg sterile zeatin (trans-isomer)                                            300 mg/l cefotaxime (Calbiochem Cat #219380)                                  50 mg/l kanamycin                                                             500X Gamborgs vitamins:                                                       5 g myo-inositol                                                              0.5 g thiamine HCL                                                            50 mg nicotinic acid                                                          50 mg pyridoxine HCl                                                          100 ml sterile water                                                          Cefotaxime is light sensitive. It turns yellow when                           it's been in the light for too long. So plates                                containing Cefotaxime were made the day before use.                           d. TM5 for root induction                                                     Ingredient           amount/liter                                             ______________________________________                                        MS salts             4.3 g                                                    Potato vitamins (200X)                                                                               5 mls                                                  Sucrose               30 g                                                    IBS (indole-3-butyric acid, Sigma)                                                                 0.1 mg (add before                                                              autoclaving)                                           Purified agar          7 g                                                    adjust pH to 5.8 with KOH                                                     Autoclave 15 minutes.                                                         When cooled to 50° C. add 25 mg kanamycin and 125 mg                   cefotaxime.                                                                   ______________________________________                                        Potato vitamins (200X)                                                        Ingredient           amount/liter                                             ______________________________________                                        myo-inositol          20 g                                                    thiamine-HCl         100 mg                                                   pyridoxine-HCl       100 mg                                                   nicotinic acid        1 g                                                     glycine              500 mg                                                   biotin                10 mg                                                   folic acid           100 mg                                                   adjust pH to 5.8 to 6.0 to clear solution.                                    Store at -20° C.                                                       ______________________________________                                    

What is claimed is:
 1. A DNA construct comprising a promotor sequenceoperably linked to a DNA sequence encoding a signal peptide from tomatoendo-1,4-β-glucanase, the DNA sequence being joined to a DNA sequenceother than a sequence encoding mature tomato endo-1,4-β-glucanase.
 2. Aplant cell comprising a DNA construct according to claim 1.